Dietary supplementation with fresh pineapple juice decreases inflammation and colonic neoplasia in IL-10-deficient mice with colitis

Abstract

Background: Bromelain, a mixture of proteolytic enzymes typically derived from pineapple stem, decreases production of proinflammatory cytokines and leukocyte homing to sites of inflammation. We previously showed that short-term oral treatment with bromelain purified from pineapple stem decreased the severity of colonic inflammation in C57BL/6 Il10(-/-) mice with chronic colitis. Since fresh pineapple fruit contains similar bromelain enzymes but at different proportions, this study aimed to determine whether long-term dietary supplementation with pineapple (supplied as juice) could decrease colon inflammation and neoplasia in Il10(-/-) mice with chronic colitis as compared with bromelain derived from stem.

Methods: Colitis was triggered in Il10(-/-) mice by exposure to the non-steroidal anti-inflammatory drug piroxicam. Mice with colitis were supplemented with fresh vs. boiled pineapple juice or bromelain purified from stem for up to 6 months.

Results: Experimental mice readily consumed fresh pineapple juice at a level that generated mean stool proteolytic activities equivalent to 14 mg bromelain purified from stem, while control mice received boiled juice with inactive enzymes. Survival was increased in the group supplemented with fresh rather than boiled juice (P = 0.01). Mice that received fresh juice also had decreased histologic colon inflammation scores and a lower incidence of inflammation-associated colonic neoplasia (35% versus 66%; P < 0.02), with fewer neoplastic lesions/colon (P = 0.05). Flow cytometric analysis of murine splenocytes exposed to fresh pineapple juice in vitro demonstrated proteolytic removal of cell surface molecules that can affect leukocyte trafficking and activation.

Conclusions: These results demonstrate that long-term dietary supplementation with fresh or unpasteurized frozen pineapple juice with proteolytically active bromelain enzymes is safe and decreases inflammation severity and the incidence and multiplicity of inflammation-associated colonic neoplasia in this commonly used murine model of inflammatory bowel disease.

Figure 1. Short-term (16 day) supplementation with fresh pineapple juice decreases severity of colitis in mice

The mean histologic score ± standard error of the mean (SEM) is shown for groups of 9 mice (* indicates p = 0.05).

Figure 2. Long-term dietary supplementation with fresh pineapple juice decreases mortality, colon inflammation, and inflammation-associated neoplasia in mice with colitis

A. Mortality in this study was due to rectal prolapse, an indicator of severe colitis in mice. 83% of mice given fresh juice survived to the 6 month study endpoint compared with 43% in mice given boiled juice with inactive enzymes (p = 0.01). B. Dietary supplementation with fresh pineapple juice for up to 26 wks also decreases colonic inflammation, with a median histologic score of 30 for mice receiving boiled juice, compared with 19 for mice receiving fresh juice (p = 0.01). C, D. Dietary supplementation with fresh pineapple juice decreased the incidence (C; p < 0.02) and multiplicity (D; p = 0.05) of inflammation-associated neoplastic lesions in the colon of Il10^−/− mice with chronic colitis.

Figure 3. Inflammation in Il10^−/− mice with colitis that were supplemented with either fresh or boiled pineapple juice for up to 6 months

Severe mucosal hyperplasia with architectural distortion and ulceration were commonly observed in the cecum (shown) and colon of mice fed boiled juice (A). Arrows point out the loss of mucosal epithelium and underlying inflammation and granulation tissue characteristic of an ulcer. Severe mucosal hyperplasia is demonstrated by the marked increase in mucosal thickness in (A) compared with mice fed fresh juice (B) who typically had only mild mucosal hyperplasia and inflammation. Panels were taken at the same magnification to emphasize the difference in mucosal hyperplasia between the 2 groups. Bar = 50 µm.

Figure 4. Neoplasia in Il10^−/− mice with colitis that were supplemented with boiled pineapple juice

Shown are examples of neoplasia in the cecum (A) and proximal colon (B) of mice fed boiled juice for up to 6 months. Arrows indicate areas of invasive adenocarcinoma. Bar = 50 µm.

Figure 5. Flow cytometric analysis of splenocytes treated in vitro with pineapple juice

A – D. Fresh pineapple juice that contains active bromelain enzymes (medium gray lines), but not boiled juice with inactive enzymes (light gray lines) specifically removes some cell surface molecules associated with leukocyte adhesion and activation (A=CD45R; B=CD62L; C= CD8; D= CD44). The reactivity of sham-treated cells was virtually super-imposable with that for boiled juice and is omitted for clarity. The black line indicates reactivity with isotype-matched control antibody. Juice was diluted 1:64 for this study. E. A summary of effects of fresh juice on cell surface molecules presents the MFI ± SEM for 3 independent experiments. Reactivity with CD45R antibody was highly sensitive to removal by fresh juice, while reactivity with CD44, CD62L, and CD8 antibodies was partially sensitive. Reactivity with CD3, CD4, and F4/80 antibodies was resistant to removal by juice.